Hi Ketul

I don't know which AAA+ ATPases you are working on, but the Rg value you got is definitely higher than all typical AAA+ ATPase/unfoldase. I calculated one Rg value from a known AAA+ ATPase, p97/VCP, which is around 56 Å. I agree that the larger Rg (13 nm) you got is apparently due to protein aggregates. To prevent the protein aggregation while SAXS measurement, the 3.5~5.0% (v/v) glycerol is usually recommended in the presence of your sample buffer. I also recommend the size exclusion chromatography (SEC) coupled with SAXS system for SAXS measurements of all the aggregation-prone protein target. Exploiting SEC-SAXS can get rid of the "interference" came from large protein aggregation and prevent a misleading Rg calculation.

Urea is a chemical denaturant that will perturb the native structure. If you focus on getting the native conformation, I won't suggest using urea to unfold the protein.

Since the molecular weight of AAA+ ATPase is relatively bigger than most of the proteins, the variable range of protein concentrations can be used from 0.5-2 mg/ml.

One more concern I'd like to draw your attention is that the radiation damage could give rise to the increase of Rg value as well, which can also be eased by adding 3.5~5.0% (v/v) glycerol in the sample buffer.

To confirm whether or not the sample is damaged, you should compare each SAXS profile at different collected time points from the beginning to the end while the data acquisition.

If you carry out SEC-SAXS and the radiation damage is happening, you will notice that the SAXS profile of each collected frame change while the protein elutes out of the column at different time points.

Frankly, SAXS is a "picky" technique that is sensitive to the buffer conditions, acquisition associated parameters (exposure time, the intensity of X-ray, temperature, ..., etc.) and intrinsically intermolecular interactions (assembly or repulsion). Just be patient and try to find out the best condition that is suitable for your system.

Best,

Yun-Tzai

Dear Dr. Lee,

thank you for your extremely valuable suggestion

these molecules are mtb AAA+ ATPase of excretion system.

the buffer contained 150 mM Nacl, 5% glycerol, 10 mM arginine.

i did not use SEC connected with SAXS but immediately collected top fraction and immediately put for SAXS.

I was wondering if the protein is forming multimers of Hexamer and ultimately its going out of limit of the SAXS machine, it showing aggregation over and again.

(limit of this Anton paar is 31.4 nm)

although I am getting a sharp peak of hexamer, this molecule undergoes multimerization on increasing concentration.

i have some Question

1> can i perform SAXS of this protein having 500mM nacl to prevent multimer formation(high salt is not recommended although but cant we subtract the buffer from protein in this case?)

2> if radiation damage is there, how to prevent it if we cant use more component in buffer

3> this protein in SEC gives single peak HEXAmer, but eventually form multimers of hexamer. how to prevent this?

is this heterogeneity in solution a reason for aggregation? does SAXS show aggregation profile for heterogeneous solution?

thanks

Dear Artur Braun

Thanks for your compliment. It's always my pleasure knowing that our knowledge and experience to really help someone.

1. 500 mM sodium chloride is fine, SAXS intensity is linearly proportional to the protein concentration, square of electron contrast of total protein relative to buffer, and protein volume. Once you increase the complexity of solutes in which is 500 mM NaCl, 5% glycerol and 10 mM arginine, the buffer subtraction become more critical than the case of the buffer with fewer solute components. Just ensure the buffer exchange complete enough.

2. You can reduce the experimental temperature or the exposure time for each SAXS frame. Reducing the exposure time will lead to loss of signal-to-noise ratio of SAXS. However this is a trade-off to prevent your sample from damage. It is better than nothing, isn't it?

3. Have you tried chemical crosslinkers with variable space lengths such as primary amine-reactive (succinimide-based, BS3) and sulfhydryl-reactive (maleimide-based, BM(PEG)2, SMCC,.. all you can find on the catalog of chemical merchandise) chemical crosslinkers to covalently stabilize the hexamer complex?

Moreover, ATP-γ-S, an ATP analog is used to stabilize hexamer conformation of typical AAA+ ATPase. You can try this out as well.

Coupling molecular stabilizers with size exclusion chromatography, I hope you can selectively "lock" the major population toward the oligomeric states you want.

As to the correlation between heterogeneity and aggregation, I have no idea how the aggregation can be induced by those species. To prevent misleading results; in my opinion, monodispersity is the only solution when you try to interpret SAXS data or use them to establish comprehensive models.

Best,

Yun-Tzai

dear Dr. Lee,

thank you very much again for highly valuable and informative prompt answer.

>If 500mM nacl is not an issue, i will definitely try this condition for SAXS

>i will also try gamma ATP ,but on which step i should use this one, from lysis buffer or SEC or after SEC.

>Regarding chemical cross linker ,i have tried glutraldehyde once for cross linking.

but i haven't tried BS3, SMCC etc.

i guess, i have to optimize the concentration of crosslinker and protein in a way that it should form only hexamer and run SEC to get hexamer peak?

> i was planing to get DLS data with different concentration of salt in solution with varying protein concentration from low to high.to get idea about in which condition protein stays as monomer.

>in case even after this condition optimization, 20% molecule stays as multimer of hexamer and 80% as only hexamer , is it possible to get saxs envelop of hexamer?

>is this oligomeric heterogeneity, a reason for aggregation profile in saxs(because i am getting only single band in sds page and sharp peak in SEC) ?

Dr. Lee , the scientific knowledge and suggestions i got from you will really be helpful for me during next SAXS data collection.

thanks